Title: Therapeutic using a bispecific antibody
United States Patent: 6,458,933
Issued: October 1, 2002
Inventors: Hansen; Hans J. (Morris Plains, NJ)
Assignee: Immunomedics, Inc. (Morris Plains, NJ)
Appl. No.: 314135
Filed: May 19, 1999
Multivalent, multispecific molecules having at least one specificity for
a pathogen and at least one specificity for the HLA class II invariant chain
(Ii) are administered to induce clearance of the pathogen. In addition to
pathogens, clearance of therapeutic or diagnostic agents, autoantibodies,
anti-graft antibodies, and other undesirable compounds may be induced using
the multivalent, multispecific molecules.
The invention relates, in general terms, to inducing clearance of a variety
of noxious substances from the body. In one aspect of the invention, there
is provided a multivalent therapeutic agent, which has at least two
different binding specificities. A representative therapeutic agent contains
at least one binding specificity for a noxious substance sought to be
cleared and at least one specificity to the HLA class II invariant chain
(Ii). In another aspect of the invention, there are provided methods of
using these therapeutic agents to induce clearance in a patient.
Due to the multiple specificities of the subject therapeutic agents, it is
likely that the inventive methods work by forming a bridge between the
agent(s) sought to be cleared from the patient and HLA class II molecules.
The resulting proximal association, in some manner, is believed to induce or
facilitate internalization of the target agent, transportation into
lysosomes and degradation of the agent therein.
As used herein "clearance" refers not only to the process of removing the
target substance from the body, but also to earlier stages of this process.
Clearance also refers to the sequestration of the target substance followed
by the removal of the target from, for example, the circulation, lymphatic
system, interstitial spaces and the body cavities.
The therapeutic agents of the invention are multivalent and multispecific.
By "multivalent" it is meant that the subject agents may bind more than one
target, which may have the same or a different structure, simultaneously. A
"target" is either the HLA class II invariant chain or an agent sought to be
cleared. By "multispecific" it is meant that the subject agents may bind
simultaneously to at least two targets which are of different structure. For
example, an agent having one specificity for HLA class II invariant chain
and one other specificity for a pathogenic bacterium would be considered
multivalent and multispecific because it can bind two structurally different
targets simultaneously. On the other hand, a molecule having two
specificities for HLA class II invariant chain, but no other specificities,
would be multivalent but not multispecific.
Some preferred agents are bispecific, but in some cases additional
specificities, e.g. two to six, are preferred. Similarly, some preferred
agents are bivalent, but increasing the valency of the agent would be
beneficial in binding either additional molecules of the same target or
multiple different targets. On preferred class of agents, therefore is
bispecific and is at least trivalent, having at least one binding site for
Ii and two for the target molecule.
As indicated above, preferred therapeutic agents have at least one
specificity directed to HLA class II invariant chain. This specificity
confers on the therapeutic agent the characteristic of targeting to
invariant chain-positive cells in many organs, such as liver, marrow,
spleen, lymph nodes and skin. This targeting is associated with rapid
clearance of the therapeutic agent containing this invariant chain
specificity through internalization, transport to lysosomes, and subsequent
The at least one other specificity of the present therapeutic agents may be
directed to nearly any substance which it is desirable to have cleared from
the body. These substances may be, for example, toxins, pathogenic organisms
(e.g., bacteria, fungi, and parasites), viruses, autoantibodies and
chemotherapeutic agents. In one embodiment, the pathogenic organism is a
fungus of the genus Cryptococcus, and especially Cryptococcus neoformans.
In another embodiment, the pathogenic organism to be cleared is a cancer
cell. The cancer cell will be bound to a phagocytic cell expressing HLA
class II invariant chain, which may be a macrophage, Kuppfer cell, or
histiocyte, for example, with the subsequent destruction of the cancer cell
by the phagocytic cell. Although the cancer cell may be internalized by the
phagocytic cell, the cancer cell may first be killed by necrotic or apoptoic
induction. The tumor-targeting moiety of the multispecific agent may be an
antibody reactive with a tumor associated or tumor specific antigen.
Another substance which is desirable to have cleared from the body is an "autoantibody,"
an antibody that recognizes a native epitope. Autoantibodies may form immune
complexes with normal cells or serum components, leading to their damage or
clearance by the immune system, just as immune complexes with foreign
pathogens. One source of tissue damage is activation of the complement
system. Most antibodies, including autoantibodies, have a site on the Fc
portion of the immunoglobulin chain that can react with activated C1q or C3
components of the complement system. Complex formation between the
autoantibody, activated C1q or C3, and the cellular surface initiates a
cascading activation of other complement components, leading to the damage
or destruction of the cell to which the autoantibody is bound.
As a result, autoantibodies are responsible for a large number of serious,
sometimes life threatening, diseases. Beeson (1994) Am. J. Med. 96:457. For
example, autoantibodies may recognize the acetylcholine receptors found in
neural muscular junctions, for example. The resulting damage to muscular
tissue leads to the development of myasthenia gravis. If the autoantibody is
directed to platelets, the resulting platelet destruction can lead to
chronic autoimmune thrombocytopenia purpura.
In one embodiment of the present invention, the at least one other
specificity of the present therapeutic agent may be directed to the specific
binding site of an autoantibody. By directing the other specificity to the
specific binding site of the autoantibody, clearance of antibodies with
potentially harmful specificities is achieved, while the clearance of
useful, normal antibodies is avoided.
In another embodiment, the present therapeutic agent may induce clearance of
autoantibodies in the form of an immune complex. In this case, the at least
one other specificity of the present therapeutic agent preferably is
directed to activated C1q, or activated C3 component, which is bound to the
immune complex. Thus, autoantibody clearance may be induced regardless of
the epitope recognized by the autoantibody specific binding site. By
directing the other specificity only to complement components involved in
immune complexes, the clearance and depletion of normal complement
components may be avoided. Antibodies that specifically recognize immune
complex-bound, activated C1q are described in U.S. Pat. No. 4,595,654,
incorporated herein by reference.
Undesirable antibodies may also have specificities against transplanted
tissue from other species. For example, porcine organs transplanted into
humans typically complex with antibodies present within the human host
within hours of transplantation. The host antibodies may activate the
complement system, resulting in damage and rejection of the transplanted
tissue, Fukushima et al. (1994) Transplantation 57:923; Pruitt et al.,
(1994) Transplantation 57:363. The major epitope on non-human tissue
responsible for transplant rejection is the (.alpha.-galactosyl epitope
(Gal.alpha.1-3Gal.beta.1-4GlcNAc-R). Galili et al. (1985) J. Exp. Med.
162:573. Up to 1% of all serum IgG in humans recognizes the .alpha.-galactosyl
epitope. Galili et al. (1984) J. Exp. Med. 160:1519.
It is one objective of the invention to ameliorate the rejection response of
humans toward non-human tissue by inducing the clearance of antibodies
directed toward transplanted tissue. In accord with this objective, the
present therapeutic agent has at least one specificity directed to the
specific binding site of an anti-graft antibody, and at least one
specificity for Ii. In a preferred embodiment, the specific binding site of
an anti-graft antibody recognizes the .alpha.-galactosyl epitope. In a more
preferred embodiment, the least one specificity for a specific binding site
of the anti-graft antibody is a polymer of alpha-galactose.
The chemical constitution of the therapeutic agents may also vary, but they
should be capable of specific binding. Accordingly, macromolecule, such as
proteins, carbohydrates (e.g., lectins) and RNAs are preferred. Due to the
well known ability to generate molecules capable of binding with a wide
range of specificities, antibodies and antibody fragments and derivatives
are particularly preferred. Both monoclonal and polyclonal antibodies may be
prepared according to established methods in the art. Because they bind with
a single, defined specificity, monoclonal antibodies are a preferred
starting material. Having generated different monoclonal antibodies, and
thus a variety different specificities, these starting molecules can be used
to generate the multivalent, multispecific agents of the invention. The art
is well versed in both recombinant and chemical methods (crosslinking) for
generating such agents.
Fragments of antibodies include any portion of the antibody which is capable
of binding the target antigen. Antibody fragments specifically include F(ab')2,
Fab, Fab' and Fv fragments. These can be generated from any class of
antibody, but typically are made from IgG or IgM. They may be made by
conventional recombinant DNA techniques or, using the classical method, by
proteolytic digestion with papain or pepsin. See CURRENT PROTOCOLS IN
IMMUNOLOGY, chapter 2, Coligan et al., eds., (John Wiley & Sons 1991-92).
F(ab')2 fragments are typically about 110 kDa (IgG) or about 150 kDa (IgM)
and contain two antigen-binding regions, joined at the hinge by disulfide
bond(s). Virtually all, if not all, of the Fc is absent in these fragments.
Fab' fragments are typically about 55 kDa (IgG) or about 75 kDa (IgM) and
can be formed, for example, by reducing the disulfide bond(s) of an F(ab')2
fragment. The resulting free sulfhydryl group(s) may be used to conveniently
conjugate Fab' fragments to other molecules, such as detection reagents
Fab fragments are monovalent and usually are about 50 kDa (from any source).
Fab fragments include the light (L) and heavy (H) chain, variable (VL
and VH, respectively) and constant (CL and CH, respectively)
regions of the antigen-binding portion of the antibody. The H and L portions
are linked by an intramolecular disulfide bridge.
Fv fragments are typically about 25 kDa (regardless of source) and contain
the variable regions of both the light and heavy chains (VL and VH,
respectively). Usually, the VL and VH chains are held together
only by non-covalent interacts and, thus, they readily dissociate. They do,
however, have the advantage of small size and they retain the same binding
properties of the larger Fab fragments. Accordingly, methods have been
developed to crosslink the VL and VH chains, using, for example,
glutaraldehyde (or other chemical crosslinkers), intermolecular disulfide
bonds (by incorporation of cysteines) and peptide linkers. The resulting Fv
is now a single chain (i.e., SCFv).
One preferred method involves the generation of SCFvs by recombinant
methods, which allows the generation of Fvs with new specificities by mixing
and matching variable chains from different antibody sources. In a typical
method, a recombinant vector would be provided which comprises the
appropriate regulatory elements driving expression of a cassette region. The
cassette region would contain a DNA encoding a peptide linker, with
convenient sites at both the 5' and 3' ends of the linker for generating
fusion proteins. The DNA encoding a variable region(s) of interest may be
cloned in the vector to form fusion proteins with the linker, thus
generating an SCFv.
In an exemplary alternative approach, DNAs encoding two Fvs may be ligated
to the DNA encoding the linker, and the resulting tripartite fusion may be
ligated directly into a conventional expression vector. The SCFv DNAs
generated any of these methods may be expressed in prokaryotic or eukaryotic
cells, depending on the vector chosen.
In one embodiment, the agent sought to be cleared is a parasite, such as an
leishmania, malaria, trypanosomiasis, babesiosis, or schistosomiasis. In
such cases the inventive molecules may be directed against a suitable
parasite-associated epitope which includes, but is not limited to, the
Parasite Epitope References
Plasmodium (NANP)3 Good et al. (1986)
Falciparum (SEQ ID NO: 1) J. Exp. Med. 164:655
(Malaria) Circumsporoz. Good et al. (1987)
protein Science 235:1059
Leishmania donovani Repetitive peptide Liew et al. (1990)
J. Exp. Med. 172:1359
Leishmani major EAEEAARLQA This application
(SEQ ID NO: 2)(code)
Toxoplasma gondii P30 surface protein Darcy et al. (1992)
J. Immunolog. 149:3636
Schistosoma mansoni Sm-28GST antigen Wolowxzuk et al. (1991)
J. Immunol 146:1987
In another embodiment, the agent sought to be cleared is a virus, such as
human immunodeficiency virus (HIV), Epstein-Barr virus (EBV), or hepatitis.
In such cases, the inventive therapeutic agent may be directed against a
suitable viral epitope including, but not limited to:
Virus Epitope Reference
HIV gp120 V3 loop, 308-331 Jatsushita, S. et al. (1988)
J Viro. 62:2107
HIV gp120 AA 428-443 Ratner et al. (1985)
HIV gp120 AA 112-124 Berzofsky et al. (1988)
HIV Reverse transcriptase Hosmalin et al. (1990)
PNAS USA 87:2344
Flu nucleoprotein Townsend et al. (1986)
AA 335-349, 366-379 Cell 44:959
Flu haemagglutinin Mills et al. (1986)
AA48-66 J. Exp. Med. 163:1477
Flu AA111-120 Hackett et al. (1983)
J. Exp. Med 158:294
Flu AA114-131 Lamb, J. and Green N. (1983)
Epstein-Barr LMP43-53 Thorley-Lawson et al. (1987)
PNAS USA 84:5384
Hepatitis B Surface Ag AA95-109; Milich et al. (1985)
AA 140-154 J. Immunol. 134:4203
Pre-S antigen Milich et al. (1986)
AA 120-132 J. Exp. Med. 164:532
Herpes simplex gD protein AA5-23 Jayaraman et al. (1993)
J. Immunol. 151:5777
gD protein AA241-260 Wyckoff et al. (1988)
Rabies glycoprotein AA32-44 MacFarlan et al. (1984)
J. Immunol. 133:2748
The agent sought to be cleared may also be bacterial. In this case, the
inventive molecule may have a specificity to a suitable bacterial epitope
which includes, but is not limited to:
Bacteria Epitope ID Reference
Tuberculosis 65Kd protein Lamb et al. (1987)
AA112-126 EMBO J. 6:1245
Staphylococcus nuclease protein Finnegan et al. (1986)
AA61-80 J. Exp. Med. 164:897
E. coli heat stable enterotoxin Cardenas et al. (1993)
Infect. Immunity 61:4629
heat liable enterotoxin Clements et al. (1986)
Infect. Immunity 53:685
Shigella sonnei form I antigen Formal et al. (1981)
Infect. Immunity 34:746
Pharmaceutical compositions according to the invention comprise at least one
therapeutic agent as described above. In addition, these compositions
typically further contain a suitable pharmaceutical excipient. Many such
excipients are known to the art and examples may be found in REMINGTON'S
PHARMACEUTICAL SCIENCES, chapters 83-92, pages 1519-1714 (Mack Publishing
Company 1990) (Remington's), which are hereby incorporated by reference. The
choice of excipient will, in general, be determined by compatibility with
the therapeutic agent(s) and the route of administration chosen. Although
the subject compositions are suitable for administration via numerous
routes, parenteral administration is generally preferred. The inventive
compositions may be formulated as a unit dose which will contain either a
therapeutically effective dose or some fraction thereof.
Methods of Preparing Multivalent Molecules
Multivalent, multispecific antibody derivatives can be prepared by a variety
of conventional procedures, ranging from glutaraldehyde linkage to more
specific linkages between functional groups. The antibodies and/or antibody
fragments are preferably covalently bound to one another, directly or
through a linker moiety, through one or more functional groups on the
antibody or fragment, e.g., amine, carboxyl, phenyl, thiol, or hydroxyl
groups. Various conventional linkers in addition to glutaraldehyde can be
used, e.g., disiocyanates, diiosothiocyanates, bis(hydroxysuccinimide)
esters, carbodiimides, maleirmidehydroxy-succinimde esters, and the like.
The optimal length of the linker may vary according to the type of target
cell. The most efficacious linker size can be determined empirically by
testing (and ensuring) reactivity to both target and Ii. Such immunochemical
techniques are well known.
A simple method to produce multivalent antibodies is to mix the antibodies
or fragments in the presence of glutaraldehyde. The initial Schiff base
linkages can be stabilized, e.g., by borohydride reduction to secondary
amines. A diiosothiocyanate or carbodiimide can be used in place of
glutaraldehyde as a non-site-specific linker.
The simplest form of a multivalent, multispecific antibody is a bispecific
antibody comprising binding specificities both to a target agent to be
cleared and to Ii. Bispecific antibodies can be made by a variety of
conventional methods, e.g., disulfide cleavage and reformation of mixtures
of whole IgG or, preferably F(ab')2 fragments, fusions of more than one
hybridoma to form polyomas that produce antibodies having more than one
specificity, and by genetic engineering. Bispecific antibodies have been
prepared by oxidative cleavage of Fab' fragments resulting from reductive
cleavage of different antibodies. This is advantageously carried out by
mixing two different F(ab')2 fragments produced by pepsin digestion of
two different antibodies, reductive cleavage to form a mixture of Fab'
fragments, followed by oxidative reformation of the disulfide linkages to
produce a mixture of F(ab')2 fragments including bispecific antibodies
containing a Fab' portion specific to each of the original epitopes (i.e.,
target and li). General techniques for the preparation of multivalent
antibodies may be found, for example, in Nisonhoff et al., Arch Biochem.
Biophys. 93: 470 (1961), Hammerling et al., J. Exp. Med. 128: 1461 (1968),
and U.S. Pat. No. 4,331,647.
More selective linkage can be achieved by using a heterobifunctional linker
such as maleimide-hydroxysuccinimide ester. Reaction of the ester with an
antibody or fragment will derivatize amine groups on the antibody or
fragment, and the derivative can then be reacted with, e.g., an antibody Fab
fragment having free sulfhydryl groups (or, a larger fragment or intact
antibody with sulfhydryl groups appended thereto by, e.g., Traut's Reagent).
Such a linker is less likely to crosslink groups in the same antibody and
improves the selectivity of the linkage.
It is advantageous to link the antibodies or fragments at sites remote from
the antigen binding sites. This can be accomplished by, e.g., linkage to
cleaved interchain sulfydryl groups, as noted above. Another method involves
reacting an antibody having an oxidized carbohydrate portion with another
antibody which has at lease one free amine function. This results in an
initial Schiff base (imine) linkage, which is preferably stabilized by
reduction to a secondary amine, e.g., by borohydride reduction, to form the
final product. Such site-specific linkages are disclosed, for small
molecules, in U.S. Pat. No. 4,671,958, and for larger addends in U.S. Pat.
The interchain disulfide bridges of the an F(ab')2 fragment having
target specificity are gently reduced with cysteine, taking care to avoid
light-heavy chain linkage, to form Fab'-SH fragments. The SH group(s) is(are)
activated with an excess of bis-maleimide linker
(1,1'-(methylenedi-4,1-phenylene)bis-malemide). An Ii-specific Mab, such as
LL1, is converted to Fab'-SH and then reacted with the activated
target-specific Fab'-SH fragment to obtain a bispecific antibody.
Alternatively, such bispecific antibodies can be produced by fusing two
hybridoma cell lines that produce anti-target Mab and anti-li Mab.
Techniques for producing tetradomas are described, for example, by Milstein
et al., Nature 305: 537 (1983) and Pohl et al., Int. J. Cancer 54: 418
Finally, such bispecific antibodies can be produced by genetic engineering.
For example, plasmids containing DNA coding for variable domains of an
anti-target Mab can be introduced into hybridomas that secrete LL1
antibodies. The resulting "transfectomas" produce bispecific antibodies that
bind target and Ii. Alternatively, chimeric genes can be designed that
encode both anti-target and anti-Ii binding domains. General techniques for
producing bispecific antibodies by genetic engineering are described, for
example, by Songsivilai et al., Biochem. Biophys. Res. Commun. 164: 271
(1989); Traunecker et al., EMBO J. 10: 3655 (1991); and Weiner et al., J.
Immunol. 147: 4035 (1991).
A higher order multivalent, multispecific molecule can be obtained by adding
various antibody components to a bispecific antibody, produced as above. For
example, a bispecific antibody can be reacted with 2-iminothiolane to
introduce one or more sulfhydryl groups for use in coupling the bispecific
antibody to an further antibody derivative that binds an the same or a
different epitope of the target antigen, using the bis-maleimide activation
procedure described above. These techniques for producing multivalent
antibodies are well known to those of skill in the art. See, for example,
U.S. Pat. No. 4,925,648, and Goldenberg, international publication No. WO
92/19273, which are incorporated by reference.
Methods of Treatment
The methods of the invention typically involve administering to a patient in
need of treatment a therapeutically effective amount of a composition which
comprises a therapeutic agent of the invention. The patient is usually
human, but may be a non-human animal. A patient will be in need of treatment
where it is desirable to induce clearance of a target agent.
A therapeutically effective amount is generally an amount sufficient to
accelerate clearance of the target agent versus a control.
Some methods involve the use of the instant therapeutic agents to induce
clearance of cytoreductive agents (chemotherapeutic agents). In a typical
method, a patient is treated with a cytoreductive agent, then the excess
cytoreductive agent is removed by administration of an inventive compound
having at least one specificity for the cytoreductive agent. In one
exemplary method, the cytoreductive agent comprises an antibody for
targeting and the inventive compound has a specificity for the antibody
portion of the agent. In this manner, any portion of the cytoreductive agent
that fails to specifically interact with its target is removed. It is
anticipated that this method will allow the use of higher, more effective
doses of the cytoreductive agent. Side effects will be minimized because the
inventive compounds induce clearance of any excess.
Other methods involve reducing the background caused by excess
non-localizing diagnostic agents. These methods are useful, for example, in
an imaging procedure where a targeting agent (e.g., an antibody) is
conjugated with a detectable marker (e.g., a radionuclide). In a typical
method, the diagnostic agent would be administered to a patient and,
following administration but before detection, an inventive compound having
at least one specificity for the diagnostic agent and at least one
specificity for Ii is provided to the patient, thereby inducing clearance of
the excess diagnostic.
Still other methods involved the clearance of pathogens, such as bacteria,
from the patient. It is envisioned that this approach will have particular
benefit for patients who have become septic. Typically, these methods
involve administering an inventive compound having at least one specificity
for a pathogen of interest (e.g., endotoxin) and at least one specificity
for Ii, thereby inducing clearance of the pathogen.
The term "treating" in its various grammatical forms in relation to the
present invention refers to preventing, curing, reversing, attenuating,
alleviating, minimizing, suppressing or halting the deleterious effects of a
disease state, disease progression, disease causative agent (e.g., bacteria
or viruses) or other abnormal condition. Because some of the inventive
methods involve the physical removal of the etiological agent, the artisan
will recognize that they are equally effective in situations where the
inventive compound is administered prior to, or simultaneous with, exposure
to the etiological agent (prophylactic treatment) and situations where the
inventive compounds are administered after (even well after) exposure to the
Claim 1 of 13 Claims
What is claimed is:
1. A multivalent molecule having at least one, specificity for a pathogenic
organism and at least one specificity for the HLA class II invariant chain
If you want to learn more
about this patent, please go directly to the U.S.
Patent and Trademark Office Web site to access the full